The present invention relates to a method for producing target protein using high-expressing peroxidase promoter (SWPA2), more particularly to a method for mass-producing target protein that comprises the steps of constructing expression vector expressing the target protein using SWPA2, transforming Agrobacteria with the expression vector, developing transformant into which the Agrobacteria is introduced and mass-producing target protein using the transformant.
Domestic (Korean) wild medicinal plants of Araliaceae family (Panax ginseng, Siberian ginseng, Dendropanax morbifera, etc) have various physiological activities such as enhancing immune response and cellular defense, anti-cancer, anti-oxidant, anti-lipid peroxidation, anti-microorganism activity, etc. Especially, Korean wild medicinal plants of Araliaceae family have much greater content of many active ingredients having excellent biological activity than any other cultivated in foreign countries.
Human lactoferrin is included in milk of human with high concentration. As a glycoprotein bound with iron, human lactoferrin has 80-kDa molecular weight and consists of two lobes. Each lobe has one iron-binding site (Metz-Boutigue et al., Eur. J. Biochem., 145, 659–676, 1984). Lactoferrin has many beneficial physiological activities and is related to various cellular defense mechanisms such as bacteriocidal and bacteriostatic action, regulation of cell proliferation, suppression of peroxy-lipid formation, regulation of immune system, regulation of iron-absorption, suppression of inflammation in infected area, anti-virus activity, prevention of E.coli from attaching to intestine cells, proliferation of Lactobacillus, etc (Yu, Korean Dairy Techno., 15, 83–89, 1997). Major products containing lactoferrin are exemplified by powdered milk for infants, cosmetics, food additives, anti-diarrhea remedies, peritoneal dialysis agents, clinical nutrition agents, hygienic products for women, medicines for the eyes, gums, etc (Nam et al., Korean J. Dairy Sci., 18, 289–298, 1996; Tomita, 1999, Morinaga Milk Industry Co., Ltd).
The production of valuable materials in plant cell cultures has been limited to plant-originated low-molecular substances taxol, shikonin, etc). But, recently, applicable area is enlarged in various fields due to the development of plant bioreactor. For example, Nitto Denko Co. produced ginseng culture cells (cultured in 20 ton tank) as a health food in Japan and Microplants Co. produced cultivated seedlings of Siberian ginseng (U.S. Pat. #09/578). In addition, the present inventors mass-produced peroxidase in sweetpotato cell cultures (Korea patent #1997-117516), isolated peroxidase genes expressed highly in culture cells (Korea patent #1998-176420) and developed high-expressing peroxidase SWPA2 promoter (PCT application #PCT/KR00/01231), leading to the development of industrial culture cell lines and the production of useful substances from the culture cells.
In regard to the production of human lactoferrin from plants or plant cell cultures, there have been reported that 48 kDa human lactoferrin-derived peptide was produced (1.8% of total water-soluble proteins) in transformed tobacco callus using CaMV 35S promoter (Mitra and Zhang, Plant Physiol., 106, 977–981, 1994), human lactoferrin was produced in tobacco plant leaves (0.3% of total protein; Salmon et al., Protein Express. Purif., 13, 127–135, 1998), in potato tuber (0.1% of total water-soluble protein) using mas P2 promoter (Chong et al., Transgenic Res., 9, 71–78, 2000), in sweetpotato callus and storage roots, potato tuber and tobacco plant (0.04–0.07% of total water-soluble protein) using CaMV 35S promoter (Liu et al., Research Report, Ministry of Science and Technology, Korea, 2000). A rice plant producing human lactoferrin (500 μg/g polished rice) by regulating rice glutelin promoter was also reported (Anzai et al., Lactoferrin: Structure, Function Appli., 265–271, 2000). However, the mass-production of human lactoferrin especially in a specific culture period using high-expressing promoter has not been reported yet.
High-expressing peroxidase promoter is a peroxidase promoter (SWPA2) induced by oxidative stress and is highly expressed in suspension culture cells of sweetpotato (PCT application #PCT/KR00/01231). The promoter showed 30-fold higher activity than CaMV 35S promoter, which was confirmed by transient assay with GUS protein using tobacco protoplasts. In addition, the promoter is expressed in tobacco plant and culture cells and especially, highly expressed in suspension culture cells of transformed tobacco in the late stage of logarithmic growth phage. The promoter is not expressed in normal plant leaves at all but is expressed when it gets oxidative stress such as ozone, low temperature, wounding, etc (Kim et al., Plant Mol. Biol., 51, 831–838, 2003).
Thus, in order to develop plant culture cells mass-producing useful protein such as human lactoferrin, the present inventors prepared plant culture cells that are available for the expression of target protein in intracellular organelles using high-expressing peroxidase SWPA2 promoter. The present inventors have completed the present invention by confirming that the target protein can be mass-produced using the prepared culture cells.